1,3-Dioxole, 2,2,4-trifluoro-5-(trifluoromethoxy)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18-Jan-2017 to 07-Mar-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 442C.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Assessed on 07-11, 14 and 16 September 2015. Dated on the 03 November 2015.

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

605-263-0

EC Number:

605-263-0

Cas Number:

161611-74-1

Molecular formula:

C4F6O3

IUPAC Name:

605-263-0

Test material form:

liquid

Details on test material:

- Physical state: Colourless liquid; odorless
- Storage condition of test material: At room temperature

Specific details on test material used for the study:

Test item preparation:
A correction factor of 1.044 was applied for the purity of the test item.
Solubility of the test item was assessed before performing the DPRA assay. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
Perfluoro methoxy dioxole stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay, 985.9 μL of cold ACN was pipetted into an amber glass vial and 14.1 μL of ice-cold Perfluoro methoxy dioxole was added. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The 100 mM test item solution in ACN was directly used for sample preparation and was kept at room temperature in a closed amber vial during this time. The test item, positive control and peptide samples were prepared less than 50 minutes before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assays.

In chemico test system

Details on the study design:

See section "Any other information on materials and methods incl. tables"

Results and discussion

Positive control results:

Cinnamic aldehyde was used as a positive control.

- Result of positive control samples in cysteine assay: Mean of SPCC depletion = 72.0% and SD = 0.8%
- Result of positive control samples in lysine assay: Mean of SPCL depletion = 43.5% and SD = 2.1%

The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

In vitro / in chemico

Results

Key result

Parameter:

other: Mean SPCC and SPCL depletion

Remarks:

(%)

Value:

45.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Reference controls

Positive controls validity:

valid

Remarks on result:

other: High reactivity

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Any other information on results incl. tables

Acceptability of the cysteine reactivity assay

The correlation coefficient (r2) of the SPCC standard calibration curve was 0.995. Since the r2 was >0.990, the SPCC standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.534 ± 0.011 mM while the mean peptide concentration of Reference Controls C was 0.534 ± 0.009 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve Perfluoro methoxy dioxole did not impact the Percent SPCC Depletion.

The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 18.33. The mean A220/A258 ratio ± 10% range was 16.50-20.16. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Acceptability of the lysine reactivity assay

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.994. Since the r2 was >0.990, the SPCL standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.492 ± 0.007 mM while the mean peptide concentration of Reference Controls C was 0.482 ± 0.007 mM.

The mean Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve Perfluoro methoxy dioxole did not impact the Percent SPCL Depletion.

The CV of the peptide areas for the nine Reference Controls B and C was 1.9%. This was within the acceptance criteria (CV <15.0%) and confirmed the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 13.98. The mean A220/A258 ratio ± 10% range was 12.58-15.38. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

SPCC and SPCL depletion and reactivity classification for the test substance

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and	Reactivity class
	Mean	+/- SD	Mean	+/- SD	SPCL depletion	Cysteine 1:10 / Lysine 1:50 prediction model
	83.4%	+/- 3.6%	8.4%	+/- 3.4%	45.9%	High reactivity

Applicant's summary and conclusion

Interpretation of results:

other: High reactivity

Conclusions:

Perfluoro methoxy dioxole was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The skin sensitisation potential of Perfluoro methoxy dioxole was evaluated using an in chemico direct peptide binding assay (DPRA) according to the OECD Guideline No. 442C and in compliance with GLP.

Following 24 hours of incubation of Perfluoro methoxy dioxole with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for Perfluoro methoxy dioxole were all within the acceptability criteria for the DPRA assay.

No co-elution of the test item with SPPC or SPCL was observed.

In the cysteine reactivity assay Perfluoro methoxy dioxole showed 83.4% SPCC depletion while in the lysine reactivity assay Perfluoro methoxy dioxole showed 8.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 45.9% and as a result Perfluoro methoxy dioxole was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, Perfluoro methoxy dioxole was considered to be positive in the DPRA.

It can be concluded that this DPRA test is valid.

Perfluoro methoxy dioxole was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.